WO2018143548A1 - Method for extracting marine collagen from fish skin - Google Patents

Abstract

The present invention relates to a method for extracting marine collagen from fish skin and, more specifically, to an extraction method capable of effectively extracting high-purity marine collagen from fish skin, which is a fish by-product to be discharged during a fish processing process. The method for extracting marine collagen from fish skin, of the present invention, comprises: a pretreatment step of removing scales and foreign matter from fish skin, which is a fish by-product; a drying step of drying the fish skin pretreated in the pretreatment step; a grinding step of obtaining a fish skin powder by grinding the fish skin dried in the drying step; a separation step of separating collagen from the fish skin powder by using an alkali and an acid; and a purification step of purifying, by using chromatography, the collagen separated in the separation step.

Description

Method of extracting marine collagen from fish

The present invention relates to a method for extracting marine collagen from fish, and more particularly, to an extraction method capable of effectively extracting high purity marine collagen from fish which is a fish byproduct discharged from a fish processing process.

Collagen is the most abundant protein in the animal body, accounting for more than 30% of body protein, and has been found to be at least 19 types (Type I-III) (Nakamura, YN et al., Relationship among collagen amount, distribution and architecture in the M. Longissimus thoracis and M. pectoralis profundus from pigs.Meat Science, 64, pp 43-50, 2003). In addition, collagen is a major protein of animal connective tissue, and supports tissues and organs and surrounds the body surface and plays a role in maintaining body shape. In particular, as a major component of connective tissue such as skin, cartilage and bone in the living body, 40% is widely distributed throughout the body, such as skin, 20% bone and cartilage, and other blood vessels and internal organs.

Unlike myofibrillar proteins, which have a double helix structure, collagen has a triple helix structure, about 14-15 nm in diameter, 280-300 nm in length, and an average molecular weight of about 300 KDa (Lehninger, AL Biochemistry, 2nd ed., Pp. 145, 1975), covalently cross-linking within the tropocollagen molecule or the tropocollagen molecule, which has a regular amino acid sequence such as (Gly-XY) n and is the basic unit molecule of the fibrous protein. crosslinking to form a physically and biologically stable structure (McClain, PE et al., Amino acid composition and cross-linking characteristics of collagen intramuscular connective tissue of striated muscle (Bos taurus) .International Journal of Biochemistry, 2 ( 7), pp 121-124, 1971).

Collagen has been applied as a raw material of leather or gelatin for a long time, and its field of application has become more diverse in recent years. In the food industry, it is used for meat packaging materials such as sausage and salami as an edible casing raw material. Collagen applied to medicines has been reported to have a healing effect on skin damaged by burns or wounds (Jeyanthi, R. et al., Solid tumour chemotherapy using implantable collagen-poly (HEMA) hydrogel containing 5-fluorouracil. of Pharmacy & Pharmacology, 43, pp. 60-62, 1991). As such, collagen is not only used as a functional material for foods and medicines, but also has a function of enhancing skin moisturizing, and thus is widely used in various fields such as basic materials of cosmetics.

In recent papers, peptides obtained from protein hydrolysates have been used as potential materials to improve wrinkles, improve moisturization, increase elasticity, and exhibit specific skin effects.

Collagen hydrolyzate, called collagen peptide, refers to a low-molecular-weight molecule in the form of peptide by hydrolyzing it through post-treatment process such as enzyme digestion after extraction of polymer collagen from pork skin and fish scales. Collagen products that have been lowered are sold.

Meanwhile, fish by-products, fishery, have been classified as food waste and used mostly as compost. Dry skin has more than 90% of protein and useful substances, so its utilization is very high, and it can be used in various ways such as collagen extraction, cosmetics, dietary supplements, and adhesives. Recently, various products such as health supplements and cosmetics using collagen are commercialized, but many depend on imports.

Increasing utilization of fish by-products in the context of Korea's per capita consumption of marine products with the world's No. 1 is very suitable for economic synergies and eco-friendly industrial development through the supply of high quality fish protein and collagen.

In addition, it contains a large amount of various polymer proteins such as collagen, albumin, myosin, elastin, and can be utilized as high-quality collagen, and it is easy to collect in domestic where the consumption of sashimi is high. As marine fish farming is also being done consistently, stable yields can be expected. Therefore, there is an urgent need to evaluate the availability of fish by-products.

(Prior art document)

(Patent literature)

1. Republic of Korea Patent No. 101020312: manufacturing method of fish scale collagen peptide

2. Republic of Korea Patent No. 101451971: Mass production method of collagen peptide derived from fish shell

The present invention has been made to improve the above problems, an object of the present invention is to provide an extraction method that can effectively extract marine collagen from the fish by-products discharged from the fish processing process.

Marine collagen extraction method from the fish of the present invention for achieving the above object comprises a pretreatment step of removing scales and foreign matter from the fish skin by-products; A drying step of drying the skin pretreated in the pretreatment step; A grinding step of grinding the dried fish in the drying step to obtain a fish powder; A separation step of separating collagen from the skin powder using an alkali and an acid; A purification step of purifying the collagen separated in the separation step by chromatography; wherein the separation step comprises the steps of: a) adding an alkali hydroxide solution to the skin powder to obtain an alkali residue from which the non-collagenic protein has been removed. And b) adding an acetic acid solution to the alkali residue, stirring and separating the supernatant with a centrifuge to extract acid-solubilized collagen, and c) adding pepsin to the acid-solubilized collagen and stirring and separating the supernatant with a centrifuge. And then dialyzing the precipitate precipitated by adding sodium chloride solution with distilled water to extract pepsin solubilized collagen.

The pretreatment step is to remove the foreign matter by washing the skin with water and then added to 0.1 to 0.2M sodium hydroxide solution and stirred for 6 to 24 hours to detach the scales attached to the skin.

The purification step is purified by ion exchange chromatography equipped with a column filled with cellulose phosphate.

In the purification step, the pepsin solubilized collagen was dialyzed at 20 mM Na ₂ HPO ₄ to inactivate pepsin and then dialyzed at 50 mM acetic acid containing 2M urea, and the fraction eluted by ion exchange chromatography was 0.5M acetic acid containing 2M NaCl. Recovered by dialysis with a solution, and then dialyzed with distilled water and lyophilized.

The purification step is purified step by step using a plurality of chromatography.

The purification step is desalting the pepsin solubilized collagen by gel filtration chromatography, followed by primary purification by ion exchange chromatography, secondary purification by hydrophobic interaction chromatography, and third purification by gel filtration chromatography.

The purification step is the first purification of the pepsin solubilized collagen by hydrophobic interaction chromatography, the second purification by ion exchange chromatography and then the third purification by gel filtration chromatography.

As described above, the present invention is to dry the skin separated from the fish at low temperature to remove the water and then processed into a powder state to extract marine collagen to prevent the deterioration of the skin and at the same time to prevent the heat denaturation of collagen and increase the extraction yield You can.

In addition, according to the present invention, a high purity marine collagen may be effectively obtained by further performing a purification process using chromatography. Marine collagen purified as described above may be useful in various applications because it maintains the collagen structure of the polymer rather than the low molecular weight collagen peptide, and has a gel forming ability and an excellent moisturizing effect.

1 and 2 are the results of measuring the ratio of acid solubilized collagen (ASC) in the skin,

Figure 3 is the result of measuring the heat denaturation temperature of acid solubilized collagen (ASC) isolated from the flounder skin,

4 to 7 are analysis results of SDS-PAGE of RS-AL and ASC extracted from fish and fish of each fish species,

8 shows the results of SDS-PAGE analysis of high purity marine collagen isolated from pepsin solubilized collagen.

Hereinafter, a method of extracting marine collagen from fish according to a preferred embodiment of the present invention will be described in detail.

Extraction method of marine collagen from the fish according to an embodiment of the present invention is a pre-treatment step of removing scales and foreign matter from the fish by-products of the fish, the drying step of drying the pre-treated skin in the pre-treatment step, and the dried fish in the drying step A pulverization step of pulverizing to obtain a skin powder, a separation step of separating collagen from the skin powder using an alkali and an acid, and a purification step of purifying the collagen separated in the separation step using chromatography. Hereinafter, look at step by step.

1. Pretreatment Step

 The pretreatment step removes scales and foreign material from the fish by-products of fish.

Fish is a shell of fish, but the shell of all fish can be applied, but preferably any one selected from flounder, rockfish, sea bass and red snapper. The four types of fish consume a lot, so it is relatively easy to supply fish. You can also use salmon skin as a fish.

Fish skin is mainly generated in the fish processing process, so to remove various foreign matters and blood powder on the fish, wash with clean water 2 to 3 times.

After washing with water may be carried out a drying step, which is the next process, but preferably removes scales attached to the skin before drying. When the pulverization process described below is performed without removing the scales, the pulverizer discharge port is clogged due to the scales, which takes a lot of time during the pulverization operation, and the pulverizer discharge parts are damaged due to the increase in the crushed material pressure due to the clogging phenomenon. There are several problems, including the phenomenon. Therefore, it is necessary to remove the scales attached to the skin. Manual removal of scales by hand using a tool can be difficult and time consuming in the process.

Therefore, the present invention uses a sodium hydroxide solution to simply and effectively remove the scales attached to the skin. For example, the skin from which the foreign matter was removed by washing with water was added to 0.1-0.2 M sodium hydroxide solution, stirred for 6 to 24 hours, and the scales attached to the skin were detached. When the fish is stirred in the sodium hydroxide solution, the scales are different depending on the type of fish, but the scales are effectively removed. In the 0.1 M sodium hydroxide solution, 80 to 90% of the scales are removed and in the 0.2 M sodium hydroxide solution, 90 to 100% of the scales are removed. Remove scales and wash again with water.

In addition, the skin is added to the sodium hydroxide solution contained in the stirring bath to reduce the scale removal rate by applying ultrasonic waves during stirring. For example, an ultrasonic vibrator may be installed at the bottom of the stirring vessel, and the stirring may be performed while applying an ultrasonic wave of 40 to 60 kHz into the stirring vessel. Ultrasonic vibration is applied to the skin while the adhesion of the scales is weakened by the sodium hydroxide solution, thereby making it easier to remove the scales.

As described above, the present invention removes the scales very easily using sodium hydroxide solution, so that there is no clogging phenomenon of the discharge part of the grinder during the pulverization of the skin, and the breakage of the grinder parts due to the clogging phenomenon does not occur. And the manual work to remove scales can be omitted, reducing manufacturing time and reducing costs and manpower.

2. Drying Step

Next, the pretreated skin is dried in the pretreatment step. The extraction yield of collagen is increased and dried to prevent deterioration and grinding. In the drying step, the skin is dried to a moisture content of 2 to 7% by weight. Freeze drying and hot air drying may be applied as a drying method. Preferably, freeze drying (FD) is used to prevent denaturation of collagen.

The freeze-drying method rapidly freezes the skin for 10 to 20 hours at a temperature of -50 to -40 ° C, and then for 48 hours at about -40 ° C in a lyophilizer having a vacuum degree of 0.1 to 0.5torr. In addition, it is a matter of course that the conventional freeze-drying method applied in the manufacture of food can be applied.

When lyophilizing the skin, moisture is removed by sublimation in the frozen state, and thus the dried product has a light porous structure, maintains its original shape and size, and is treated at low temperature without applying heat. Transfer of soluble components, non-enzymatic browning and protein denaturation rarely occur in drying.

In the case of hot air drying, the skin may be dried by hot air at 30 to 80 ° C. In order to prevent denaturation of collagen during hot air drying, the temperature of hot air is 65 ° C. or lower, preferably 40 to 60 ° C. Collagen contained in the skin has a higher denaturation temperature than the separated state.

3. Crushing Step

The dried fish is ground to a suitable size using a grinder to obtain the fish powder. For example, it can be ground to a size of 50 to 150 mesh particle size.

4. Separation step

After the skin powder is obtained, collagen is separated from the skin powder.

For example, the separation step may include a) adding an alkali hydroxide solution to the skin powder to obtain an alkali residue from which the non-collagenic protein has been removed, and b) adding an acetic acid solution to the alkali residue, stirring and separating the supernatant with a centrifuge. Extracting the acid-solubilized collagen, and c) adding pepsin to the acid-solubilized collagen, stirring and separating the supernatant with a centrifugal separator, and then adding the sodium chloride solution to dialyzate the precipitate precipitated with distilled water to extract the pepsin solubilized collagen. Is made of.

The step of obtaining an alkali residue from which non-collagenic protein was removed by adding sodium hydroxide solution to skin powder is as follows.

The skin powder and sodium hydroxide solution were mixed at a weight ratio of 1: 5 to 10, and then stirred at room temperature (20 to 25 ° C.) for 12 to 24 hours to obtain an alkali residue from which non-collagenic protein was removed using a centrifuge. can do.

In addition, the step of extracting the acid-solubilized collagen by adding acetic acid solution to the alkali residues is as follows.

Alkaline residue is washed with distilled water and acetic acid is added to extract collagen. Alkaline residue and acetic acid solution are mixed at a weight ratio of 1: 5 to 10, and then stirred at room temperature (20 to 25 ° C) for 12 to 24 hours, and the supernatant is separated using a centrifuge to obtain acid solubilized collagen.

In addition, the step of extracting pepsin solubilized collagen by adding pepsin to the acid solubilized collagen is as follows.

Pepsin is added to the acid-solubilized collagen, stirred for 10 to 20 hours, the supernatant is separated by centrifugation, and the precipitate precipitated by adding 2M sodium chloride solution is dialyzed with distilled water to obtain pepsin solubilized collagen.

5. Purification step

Pepsin solubilized collagen isolated in the separation step may be purified using chromatography to obtain high purity marine collagen.

As an example, the purification step may be purified using ion exchange chromatography equipped with a column packed with cellulose phosphate.

Pepsin solubilized collagen was dialyzed in 20 mM Na ₂ HPO ₄ to inactivate pepsin, then dialyzed against 50 mM acetic acid solution (pH 4.8) containing 2M urea, and then fractions were eluted using ion-exchange chromatography. Let's do it. For example, purification was performed with a linear gradient (60 ml / h) of 0-600 mM NaCl in a column filled with cellulose phosphate (P11, Whatman, Maidstone, UK), and the fraction eluted at 230 nm was 0.5 M acetic acid containing 2.0 M NaCl. The solution is recovered by dialysis and then dialyzed with distilled water and lyophilized to obtain high purity marine collagen.

On the other hand, as another example of the purification process can be purified step by step using a plurality of chromatography. Examples of the chromatography that can be used include ion-exchange chromatography, gel filtration chromatography, and hydrophobic interaction chromatography.

For example, desalination of pepsin solubilized collagen by gel filtration chromatography, followed by primary purification by ion exchange chromatography, secondary purification by hydrophobic interaction chromatography, and third purification by gel filtration chromatography. Can be purified.

In another example, collagen may be purified by first purifying the pepsin solubilized collagen by hydrophobic interaction chromatography, secondarily purifying by ion exchange chromatography, and third purifying by gel filtration chromatography.

Hereinafter, the content of the present invention will be described in detail through the following experimental examples. This is for explaining the present invention in more detail, and the scope of the present invention is not limited to the following experimental examples.

<Example>

After washing the skins of the four species of fish (Urug, perch, flounder, red snapper), the scales were removed and then rapidly frozen at -45 ° C for 15 hours and then for 48 hours at -40 ° C in a freeze dryer with a vacuum of 0.5torr. After drying, it was ground to prepare a fish powder. 0.1 M sodium hydroxide solution was added to the fish powder at a 10-fold weight ratio, and the mixture was stirred at room temperature (20 ° C.) for 16 hours, and then an alkali residue (RS-AL) from which the non-collagenic protein was removed was removed using a centrifuge. Obtained.

The obtained alkaline residue was washed with distilled water, 0.5M acetic acid solution was mixed at a weight ratio of 10 times, stirred at room temperature (20 ° C) for 16 hours, and the supernatant was separated using a centrifuge to obtain acid solubilized collagen (ASC). ) Was obtained. Pepsin (EC 3.4.23.1; crystallized and lyophilized, Sigma, MO) was added to acid-solubilized collagen, stirred for 14 hours, the supernatant was separated by centrifugation, and the precipitate precipitated by adding 2M sodium chloride solution was dialyzed with distilled water. Pepsin solubilized collagen was obtained.

Pepsin solubilized collagen was dialyzed in 20mM Na ₂ HPO ₄ to inactivate pepsin and then dialyzed in 50mM acetic acid solution (pH 4.8) containing 2M urea, followed by ion chromatography (P11, Whatman, Maidstone, UK). Purification was carried out in a linear gradient (60ml / h) of 0 ~ 600mM NaCl in a column packed with, and the fraction eluted at 230nm was recovered by dialysis with 0.5M acetic acid solution containing 2.0M NaCl, and then dialyzed with distilled water and freeze-dried. The high purity marine collagen was obtained.

1. Investigation of the ratio of acid soluble collagen (ASC) in the skin

In order to investigate the ratio of ASC obtained from fish to each fish species, 1 ml of dialysis-completed ASC was put into 1.5 ml tube, lyophilized and weighed, and weighed and dialyzed before and after freeze drying in 1.5 ml tube. The ratio of the ASC extracted was examined, and the ratio of ASC extracted in the skin was calculated as follows.

S = {(Y1 × Y2) / X} × 100

S:% ASC in fish

Y1: weight of salt precipitate before dialysis (g)

Y2:% ASC to dialysis lyophilized gel

X: wet weight of the first used skin (g)

The experimental results are shown in FIG. 1.

Referring to Figure 1, the results of the survey of the acid-soluble collagen (ASC) ratio of flounder, rockfish, sea bass, red snapper showed the highest in the wet weight of red snapper 9.88%, flounder 8.01 ± 0.19%, perch was 5.75% The lowest value was 2.69%.

When the collagen content of four tibiae fishes was compared, it showed a tendency to support histological observations by showing a large difference by fish species, but the relative value was very low.

The ratio of ASC collagen in the skin was analyzed using Sircol ^™ Soluble Collagen Assay kit (Biocolor, UK). The calibration curve of the standard solution was prepared, and the collagen content of the sample to be analyzed was obtained and shown in FIG. 2.

The collagen content in the skin was measured using the Collagen Assay kit, and the flounder was 9.73%, Uru 3.46%, perch 6.96%, and red snapper 11.83%.

2. Investigation of the collagen thermophilic stability

The heat denaturation temperature was measured at a constant heating rate (0.5 ° C./1 min) for acid-solubilized collagen isolated from flounder skin using Micro DSC (Setaram, France). At this time, rat tail tendon ASC of land vertebrate was purchased. The heat denaturation temperature was measured by the same method and compared with the data of the fish, and the results are shown in FIG. 3.

Thermal denaturation temperature was measured at a constant heating rate (0.5 ℃ / 1 minute) using a Micro DSC (Setaram, France), and at the same time, the tail tendon ASC (Sigma Aldrich, USA) of terrestrial vertebrates was purchased as a control. As a result of measuring the heat denaturation temperature and comparing with the data of the fish, fish collagen (Fig. 3: ○, ●) was 12 ℃ lower than rat collagen (Fig. 3: ▲), a terrestrial vertebrate.

The low denaturation temperature suggested that fish collagen had a low degree of proline hydroxylation. In addition, flounder skin ASC showed higher resistance to heat denaturation due to higher heat degeneration temperature of 3.9 ° C than rainbow trout muscle ASC.

3. Molecular Characterization

The molecular characteristics of collagen were analyzed by constituent amino acid investigation and SDS-PAGE analysis.

The constituent amino acid was weighed 0.5g of acid-solubilized collagen into an 18ml test tube, 3ml of 6N HCl was added, and the test tube was sealed using a vacuum pump. The sealed test tube was hydrolyzed at a heating block at 121 ° C. for 24 hours, and then, after removing the acid with a rotary evaporator at 50 ° C. and 40 psi, 10 ml of sodium loading buffer was added, followed by 1 ㎖ was filtered off with a membrane filter (0.2 μl) and quantitated with an amino acid analyzer (S-433H, SYKAM GmbH, Germany). As the analysis conditions, a cation separation column (LCA K06 / Na) was used as the column, the column size was 4.6 × 150 mm, the column temperature was 57-74 ° C, the buffer flow rate was 0.45 ml / min, and the reagent flow rate was 0.25. The pH range was 3.45 ~ 10.85 and the wavelength was 440nm and 570nm.

In order to confirm protein molecular weight, SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electorphoresis) was performed according to the method of Laemmli (1970).

Each sample was prepared at a concentration of 1 mg / ml in sample buffer (50 mM Tris-HCL, pH 7.5; 50% glycerin, 1% SDS, 0.02% bromophenol blue, BPB), and then heated and denatured at 95 ° C. for 5 minutes. Samples were prepared by cooling at room temperature for 10 minutes. The prepared sample was prepared by using a 40% polyacrylamide composed of 3% stacking gel and 7.5% separate gel to prepare 7.5% gel. The electrophoretic apparatus was 200V, using Bio-RAD Power Pac Basic (USA). It carried out on the conditions of 35 mA / gel. Protein band staining was prepared according to Fairbanks et al., (1971) in four stages of dyeing solution of Coomassie brilliant blue (CBB), 2-propanol and acetic acid in stages. 2 hours each. The marker used to confirm the molecular weight of the sample was SDS-PAGE Molecular Weight Stadards (Bio-rad Laboratirories, High range, USA).

SDS-PAGE results for each fish species are shown in FIGS. 4 to 7, respectively. Figure 4 is a SDS-PAGE pattern of RS-AL, ASC extracted from flounder and flatfish, Figure 5 is a SDS-PAGE pattern of RS-AL, ASC extracted from rock and rock, Figure 6 is a perch fish and SDS-PAGE patterns of RS-AL and ASC extracted from sea bass, and FIG. 7 shows SDS-PAGE patterns of RS-AL and ASC extracted from red snapper and red snapper. In Figures 4 to 7 MP refers to a marker protein (maker protein).

As a result of SDS-PAGE for each species, all four species consisted of subunit structures α1 (I), α2 (I) and dimers of β-chain, and two polymer bands were identified above β-chain. All of them showed the SDS-PAGE pattern of typical type I collagen, and the molecular weights of α1 (I) and α2 (I) were slightly above those of 116.2 k in fish, RS-AL, and ASC. In the vicinity of 200k, β-chain protein bands were predominantly distributed.

Constituent amino acid analysis results are shown in Table 1 below.

Amino Acid (mg / 100g) Flounder Rockfish Perch True dome Asp 4.31 4.75 4.41 4.02 Thr 1.94 1.91 2.31 1.93 Ser 3.32 4.07 3.12 2.85 Glu 7.27 7.36 7.56 7.01 Pro 10.86 10.53 12.46 10.50 Gly 17.64 17.52 18.37 17.20 Ala 7.02 6.63 7.62 7.13 Cys 0.05 0.06 0.05 0.04 Met 1.74 1.90 1.68 1.52 Val 1.35 1.33 1.49 1.38 Ile 0.80 0.72 0.74 0.71 Leu 1.85 1.71 1.83 1.80 Tyr 0.27 0.15 0.33 0.37 Phe 1.69 1.71 1.64 1.42 His 0.76 0.80 0.78 0.70 Lys 2.68 2.58 2.61 2.45 Amo 1.21 1.36 1.07 1.02 Arg 5.38 5.58 5.93 5.37 Total 70.15 70.67 74.02 67.41

Table 1 shows the constituent amino acid results of acid-soluble collagen (ASC) extracted from the fish of the flounder, rockfish, sea bass, red snapper.

The total constituent amino acids of the flounder, urchin, perch and red snapper fish ASC were 70.15g / 100g, 70.67g / 100g, 74.02g / 100g and 67.41g / 100g. Glycine, which can identify the characteristics of the repeated Gly-XY amino acid sequence, accounted for about 25% (17.64g / 100g, 17.52g / 100g, 18.37g / 100g, 17.20g / 100g, respectively), and proline 10.86g / 100g 10.53g / 100g, 12.46g / 100g and 10.50g / 100g accounted for about 15%.

4. Analysis of high purity marine collagen

SDS-PAGE analysis of high purity marine collagen isolated from pepsin solubilized collagen is shown in FIG. Collagen was detected in high-purity marine collagen of four fish. Collagen was composed of α1 (I) and α2 (I) subunits such as ASC and β-chain as dimers, and two polymer bands were identified above β-chain. MP: marker protein in FIG. 11; A: flounder; B: uruk; C: perch; D: It means red snapper.

Unlike other proteins, collagen contains hydroxylated amino acids, hydroxyproline and hydroxylysine, and consists of repeating amino acid sequence of Gly-XY in the triple helix region, where proline and hydroxyproline are located at X and Y positions. Frequently located, the presence of collagen can be determined by glycine, proline and hydroxyproline content.

Table 2 shows the results of analyzing the amino acid residues to determine the content of glycine, proline, hydroxyproline, and the like for high purity marine collagen.

Amino Acid (mg / 100g) residues (%) Hydroxyproline 75 8% Asp 36 4% Thr 36 4% Ser 46 5% Glu 77 8% Pro 84 8% Gly 353 35% Ala 114 11% Half-cys 0 0% Val 25 3% Met 9 One% Ile 17 2% Leu 22 2% Tyr 2 0% Phe 12 One% Hydroxylysine 9 One% Lys 25 3% Histidine 8 One% Arg 50 5% Total 1,000 100%

Referring to Table 2, the amino acid residues were analyzed to determine the content of glycine, proline, hydroxyproline, etc., for high purity marine collagen. 8%) and hydroxylysine 9 (1%).

As described above, the present invention has been described with reference to one embodiment, which is merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent embodiments are possible.

Claims (7)

A pretreatment step of removing scales and foreign substances from fish which is a fish byproduct; A drying step of drying the skin pretreated in the pretreatment step; A grinding step of grinding the dried fish in the drying step to obtain a fish powder; A separation step of separating collagen from the skin powder using an alkali and an acid; And a purification step of purifying the collagen separated in the separation step using chromatography. The separation step is a) adding a sodium hydroxide solution to the skin powder to obtain an alkali residue from which the non-collagenic protein is removed, b) adding an acetic acid solution to the alkali residue and stirring, and then separating the supernatant with a centrifuge Extracting acid-solubilized collagen, c) adding pepsin to the acid-solubilized collagen, stirring, separating the supernatant with a centrifugal separator, and then adding sodium chloride solution to dialyzate the precipitate precipitated with distilled water to extract pepsin-solubilized collagen. A method for extracting marine collagen from fish, comprising: The method of claim 1, wherein the pretreatment step is to remove the foreign matter by washing the skin with water and then added to 0.1 to 0.2M sodium hydroxide solution and stirred for 6 to 24 hours to detach the scales attached to the skin Extraction method of marine collagen from fish. The method of claim 1, wherein the purification step is performed by ion exchange chromatography equipped with a column filled with cellulose phosphate. 4. The method of claim 3 wherein the purification step and then dialyzed in 50mM acetic acid containing the following 2M element that inactivate the pepsin by dialysis the pepsin solubilized collagen in a 20mM Na ₂ HPO ₄ fraction eluted with the ion exchange chromatography 2M A method of extracting marine collagen from fish, characterized in that it is recovered by dialysis with 0.5M acetic acid solution containing NaCl and then lyophilized after dialysis with distilled water. The method of claim 1, wherein the purification step is to extract marine collagen from the skin, characterized in that the purification step by using a plurality of chromatography. According to claim 5, wherein the purification step desalting the pepsin solubilized collagen by gel filtration chromatography, the first purification by ion exchange chromatography, the second purification by hydrophobic interaction chromatography and then by gel filtration chromatography 3 A method of extracting marine collagen from fish, characterized in that it is purified by tea. The method of claim 5, wherein the purification step is the first step of purifying the pepsin solubilized collagen by hydrophobic interaction chromatography, second purification by ion exchange chromatography and third purification by gel filtration chromatography Method of extracting marine collagen from.

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